The plant kingdom produces tens of thousands of different small compounds with very complex structures that are often genus or family specific. These molecules, referred to as “secondary metabolites” or “specialized metabolites,” display an immense variety in structures and biological activities that plants have tapped into over the course of evolution, and that is now harnessed by man for industrial and medical applications. These compounds play, for example, a role in the resistance against pests and diseases, attraction of pollinators and interaction with symbiotic microorganisms. Besides the importance for the plant itself, secondary metabolites are of great interest because they determine the quality of food (color, taste, aroma) and ornamental plants (flower color, fragrance). A number of secondary metabolites isolated from plants are commercially available as fine chemicals, for example, drugs, dyes, flavors, fragrances and even pesticides. In addition, various health-improving effects and disease-preventing activities of secondary metabolites have been discovered. Flavonoids and terpenoids, for example, have health-promoting activities as food ingredients, and several alkaloids have pharmacological activities. To illustrate this further, taxol is a highly substituted, polyoxygenated cyclic diterpenoid characterized by the taxane ring system, which presents an excellent anti-tumoral activity against a range of cancers.
Although about 100,000 plant secondary metabolites are already known, only a small percentage of all plants have been studied to some extent for the presence of secondary metabolites. Interest in such metabolites is growing as, e.g., plant sources of new and useful drugs are discovered. Some of these valuable phytochemicals are quite expensive because they are only produced at extremely low levels in plants. In fact, very little is known about the biosynthesis of secondary metabolites in plants. However, some recently elucidated biosynthetic pathways of secondary metabolites are long and complicated, requiring multiple enzymatic steps to produce the desired end product. Most often, the alternative of producing these secondary metabolites through chemical synthesis is complicated due to a large number of asymmetric carbons and, in most cases, chemical synthesis is not economically feasible.
The cellular and genetic programs that steer the production of secondary metabolites can be launched rapidly when plants perceive particular environmental stimuli. The jasmonate phytohormones (JAs) play a prominent and universal role in mediating these responses as they can induce synthetic pathways of molecules of a wide structural variety, encompassing all major secondary metabolites (Zhao et al. 2005; Pauwels et al. 2009). Essential in the “core JA signaling module” in plants is the F-box protein CORONATINE INSENSITIVE 1 (COI1), which is part of a Skp/Cullin/F-box-type E3 ubiquitin ligase complex (SCFCOI1), to which it provides substrate specificity. The targets of the SCFCOI1 complex are the JA ZIM domain (JAZ) family of repressor proteins. JAZ and COI1 proteins directly interact in the presence of the bioactive JA-isoleucine (JA-Ile) conjugate to form a co-receptor complex, which triggers the degradation of the JAZ proteins by the 26S proteasome. The JAZ proteins are further characterized by a conserved C-terminal JAs domain, which is required for the interaction with both COI1 and a broad array of transcription factors (TFs). JA-triggered JAZ degradation releases these TFs, which each modulate expression of specific sets of JA-responsive genes and, thereby, the production of specific sets of secondary metabolites (De Geyter et al. 2012, Trends Plant Sci. 17:349-359).
In Arabidopsis thaliana, for example, the basic helix-loop-helix (bHLH) factor MYC2 is the best known target of the JAZ proteins. MYC2 has been shown to be both directly and indirectly involved in regulating secondary metabolite induction, more precisely, of phenolic compounds and glucosinolates. The Catharanthus roseus MYC2 homologue regulates the expression of the ORCA TFs by direct binding to the “on/off switch” in the promoter of the ORCA3 gene, and thereby controlling expression of several alkaloid biosynthesis genes. In Nicotiana tabacum, MYC2 proteins up-regulate the ORCA-related NIC2 locus APETALA2/ETHYLENE Response Factor (AP2/ERF) TFs that regulate nicotine biosynthesis as well as the nicotine biosynthesis enzymes themselves. JAZ proteins also directly interact with and thereby repress other TFs with a well-established role in the synthesis of secondary metabolites, such as the bHLH TFs GLABRA3 (GL3), ENHANCER OF GL3 (EGL3) and TRANSPARENT TESTA8 (TT8), and the R2R3-MYB TF PAP1, which together compose transcriptional activator complexes that control anthocyanin biosynthesis and are conserved in the plant kingdom.
Besides direct JAZ interactors, other TFs with a proven role in JA-mediated elicitation of a specific metabolic pathway exist, such as WRKY-type TFs that regulate sesquiterpene biosynthesis in various plants, but the full picture on how the central module exerts control over evolutionary distant metabolic pathways, leading to natural products of a wide structural variety, is still lacking. Although overexpression of several of these transcription factors could stimulate synthesis of some secondary metabolites, no master switches have been found that can mimic the full JA spectrum, neither quantitatively nor qualitatively, or replace JAs in plant engineering programs. Likewise, for many secondary metabolic pathways, such as of triterpenes, no regulatory TFs have been identified yet.
Therefore, a need exists for novel ways, preferably generic ways, to regulate the production of secondary metabolites in plants and plant-derived cell cultures.